Insights into the Role of the Peroxisomal Ubiquitination Machinery in Pex13p Degradation in the Yeast Hansenula polymorpha

Abstract

The import of matrix proteins into peroxisomes in yeast requires the action of the ubiquitin-conjugating enzyme Pex4p and a complex consisting of the ubiquitin E3 ligases Pex2p, Pex10p and Pex12p. Together, this peroxisomal ubiquitination machinery is thought to ubiquitinate the cycling receptor protein Pex5p and members of the Pex20p family of co-receptors, a modification that is required for receptor recycling. However, recent reports have demonstrated that this machinery plays a role in additional peroxisome-associated processes. Hence, our understanding of the function of these proteins in peroxisome biology is still incomplete. Here, we identify a role for the peroxisomal ubiquitination machinery in the degradation of the peroxisomal membrane protein Pex13p. Our data demonstrate that Pex13p levels build up in cells lacking members of this machinery and also establish that Pex13p undergoes rapid degradation in wild-type cells. Furthermore, we show that Pex13p is ubiquitinated in wild-type cells and also establish that Pex13p ubiquitination is reduced in cells lacking a functional peroxisomal E3 ligase complex. Finally, deletion of PEX2 causes Pex13p to build up at the peroxisomal membrane. Taken together, our data provide further evidence that the role of the peroxisomal ubiquitination machinery in peroxisome biology goes much deeper than receptor recycling alone.

Peroxisome biogenesis, membrane contact sites, and quality control

“…Last, but not the least, PMPs in the peroxisome membrane could also be misfolded or mis‐assembled and subject to turnover. Indeed, in H. polymorpha , Pex13 is subject to ubiquitination and degradation . This process is dependent both on Ub and the peroxisomal ubiquitination machinery (the E2 enzyme, Pex4, and the RING E3 complex component, Pex2), but a direct involvement of the ubiquitination machinery in Pex13 ubiquitination is still missing.…”

Post‐translational modifications of proteins associated with yeast peroxisome membrane: An essential mode of regulatory mechanism

“…This finding was further supported by the observation that deletion of the components of ubiquitination machinery resulted in increased levels of Pex13 (Chen et al, 2018). A significant increase in Pex13 was observed in cells lacking Pex2 compared to WT cells (Chen et al, 2018). An increased level of Pex13 was also observed in pex5, pex8 and pex14 cells.…”

“…As mentioned earlier, it forms a docking complex along with Pex14 and Pex17 required for the import of matrix proteins (Erdmann & Schliebs, 2005). A recent study in O. polymorpha reported ubiquitin-mediated degradation of Pex13 (Chen et al, 2018). Pex13 is monoubiquitinated by the E2 Pex4 and degraded via the proteasome by the peroxisomal E3 ligase complex (Pex2, Pex10 and Pex12) (Chen et al, 2018).…”

Peroxisome retention involves Inp1-dependent peroxisome–plasma membrane contact sites in yeast

“…The PCR fragment was digested with Bpu10I and NotI, and ligated in Bpu10I and NotI digested plasmid pHIPZ Inp1-mKate2, resulting in plasmid pHIPN Inp1-mKate2. Plasmid pHIPZ Inp1-mKate2 was obtained as follows: the fragment encoding the C terminus of the INP1 gene was digested with BglII and HindIII from plasmid pHIPZ Inp1-GFP (pAMK6, Krikken et al, 2009), then inserted between the BglII and HindIII sites of pHIPZ Pex14-mKate2 (Chen et al, 2018), producing pHIPZ Inp1-mKate2.…”

“…Plasmid pAMK142 was constructed as follows: a PCR fragment containing PMP47 was amplified with primers PMP47_fw and PMP47_rev using WT genomic DNA as a template. The obtained PCR fragment was digested with BamHI and HindIII, and inserted between the BglII and HindIII sites of pHIPZ Pex14-mKate2 (Chen et al, 2018), resulting in plasmid pAMK142.…”